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T K Das

Publications and source records attributed to T K Das.

At least 19 recordsLinked to original sources
PubMed2004

Novel mode of resistance to Fusarium infection by a mild dose pre-exposure of cadmium in wheat.

Exposure of healthy wheat seeds (Triticum aestivum var Sonalika) to mild dose of cadmium (Cd(2+)) given as 50 microM CdCl(2) for 48 h and then washed off Cd(2+) offered resistance to the subsequent infection by Fusarium oxysporum inoculum. Seven days old seedlings having two primary leaves were aseptically inoculated with fungus, F. oxysporum (1 x 10(6)) spores. The seedlings pre-exposed to low level of Cd(2+) survived the Fusarium infection, while plantlets without Cd(2+) stress wilted and then perished due to Fusarium infection. The stress associated proteins induced by Cd(2+) (50 microM), F. oxysporum and by the co-stress (50 microM Cd(2+) and then with F. oxysporum) treatments were observed to be of same molecular weight (51 kDa). Antibody was raised against the purified Cd(2+)-stress associated protein (CSAP). Immuno-gold labeling of wheat seedling root tissue showed the presence of this CSAP in Cd(2+) pre-exposed and in co-stressed tissues and to be located predominantly on the inner linings of the cell membranes. We also observed that the anti-CSAP-antibody also labeled the root tissue of only Fusarium inoculated seedlings and the gold labeling was intensely located on the membrane. This cross-reaction of anti-CSAP suggests that Fusarium-induced stress protein (FISP) possibly has close homology to CSAP. We thus show for the first time the over expression of a high molecular mass protein by mild dose of Cd(2+) pre-exposure to wheat seeds which subsequently provided protection against Fusarium infection. This mode of resistance developed by an abiotic stress-causing agent against pathogen infection is novel.

Blotting, Western↗
PubMed2003

Chronic oral administration of raw garlic protects against isoproterenol-induced myocardial necrosis in rat.

Wistar albino rats (150-200 g) were fed raw garlic homogenate orally in three different doses (125, 250, 500 mg/kg/day) for 30 days. Isoproterenol (85 mg/kg, s.c. 2 doses at 24-h interval, animals sacrificed after 24 h of last injection) induced myocardial necrosis in control rats and after 30 days of garlic feeding. Myocardial oxidative stress was evident following isoproterenol administration by reduction in myocardial superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities along with a rise in plasma thiobarbituric acid reactive substances (TBARS). Myocardial necrosis was evident from the light microscopic and ultrastructural changes, along with a rise in plasma lactate dehydrogenase (LDH). Significant preservation of myocardial SOD activity was observed in all the garlic-fed rats. However, there was no significant change in myocardial reduced glutathione level and GPx activity in any of the treated groups. Significant reduction in plasma TBARS and LDH levels was observed in the 500 mg/kg garlic treated group. Isoproterenol-induced myocardial morphological changes were least in the 250 and 500 mg/kg garlic treated groups. The results suggest that chronic oral administration of raw garlic offered protection against isoproterenol-induced myocardial necrosis and associated oxidative stress.

Animals↗
PubMed2002

Detection of root mucilage using an anti-fucose antibody.

Plant root mucilage is known to enhance soil quality by contributing towards the soil carbon pool, soil aggregation, detoxification of heavy metal ions and interactions with rhizospheric microflora. Mucilage consists of many monosaccharide units, including fucose which can be used as an indicator for plant root based polysaccharides. This is the first report of an immunological technique developed to use anti-fucose antibodies as markers for probing and localizing fucosyl residues in mucilage polysaccharide and, in turn, for localization of plant root mucilage. Fucose was complexed with bovine serum albumin to raise antibodies against fucose. A fucose-directed antibody was shown to cross-react with root cap mucilages from grasses. This antibody was used to localize root mucilage polysaccharide in maize and wheat root caps using immunogold electron microscopy. Abundant labelling could be localized on the cell wall, and in the intercellular matrix and vesicles of the peripheral root cap cells. Labelling was less intense in cells towards the centre of the root cap tissue. Control experiments confirmed that immunogold localization of fucose was specific and reliable.

Animals↗
PubMed2001

Binding of NO and CO to the d(1) Heme of cd(1) nitrite reductase from Pseudomonas aeruginosa.

The cd(1) nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide. The enzyme contains two redox centers, a c-type heme and a unique d(1) heme, which is a dioxoisobacteriochlorin. Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d(1) cofactor with extremely high affinity and inhibit enzyme activity. In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d(1) site of the reduced enzyme. The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heme complexes. The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm(-1), respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm(-1), respectively, for the CO complex. Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heme proteins. The unusual spectroscopic characteristics of the d(1) cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand. It is likely that the influence of a highly ruffled structure of heme d(1) on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies.

Carbon Monoxide↗
PubMed2001

Simultaneous observation of the O---O and Fe---O2 stretching modes in oxyhemoglobins.

Understanding of the chemical nature of the dioxygen moiety of oxyhemoglobin is crucial for elucidation of its physiological function. In the present work, direct Raman spectroscopic observation of both the FeO(2) and OO stretching modes unambiguously establishes the vibrational characteristics of the oxygen-bound heme moiety in the hemoglobins of Chlamydomonas eugametos and Synechocystis PCC6803. In addition to providing the resonance Raman assignment of the OO stretching mode (1136 cm(-1) for Chlamydomonas, 1133 cm(-1) for Synechocystis) in an oxyhemoglobin with an iron-porphyrin, this study also reports unusually low frequencies for the FeO(2) stretching modes (554 cm(-1)). The effect of strong hydrogen bonding to the bound oxygen is confirmed by changes in the frequency of the FeO(2) stretching mode on mutation of distal residues. These findings suggest an enzymatic function rather than an oxygen transport role for these hemoglobins.

Animals↗
PubMed2001

pH-dependent structural changes at the Heme-Copper binuclear center of cytochrome c oxidase.

The resonance Raman spectra of the aa3 cytochrome c oxidase from Rhodobacter sphaeroides reveal pH-dependent structural changes in the binuclear site at room temperature. The binuclear site, which is the catalytic center of the enzyme, possesses two conformations at neutral pH, assessed from their distinctly different Fe-CO stretching modes in the resonance Raman spectra of the CO complex of the fully reduced enzyme. The two conformations (alpha and beta) interconvert reversibly in the pH 6-9 range with a pKa of 7.4, consistent with Fourier transform infrared spectroscopy measurements done at cryogenic temperatures (D.M. Mitchell, J.P. Sapleigh, A.M.Archer, J.O. Alben, and R.B.Gennis, 1996, Biochemistry 35:9446-9450). It is postulated that the different structures result from a change in the position of the Cu(B) atom with respect to the CO due to the presence of one or more ionizable groups in the vicinity of the binuclear center. The conserved tyrosine residue (Tyr-288 in R. sphaeroides, Tyr-244 in the bovine enzyme) that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu(B) are possible candidates. The existence of an equilibrium between the two conformers at physiological pH and room temperature suggests that the conformers may be functionally involved in enzymatic activity.

Animals↗
PubMed2001

Garlic-induced alteration in rat liver and kidney morphology and associated changes in endogenous antioxidant status.

The effects of chronic garlic intake on various endogenous antioxidant enzymes and lipid peroxidation on two major organs, the liver (L) and kidneys (K), were investigated. Wistar albino rats were fed with fresh garlic homogenate daily by gavage in three different doses (250, 500 and 1000 mg/kg/day) for 30 days. After this period, rats were sacrificed and liver and kidneys were harvested for biochemical estimation. In comparison to saline-treated rats, the 250 mg/kg/day dose significantly (P<0.02) reduced thiobarbituric acid reactive substances (TBARS) (L: 187.48+/-9.23 vs 150.66+/-11.45; K: 177.38 15.88 vs 120.66+/-9.39 nmol/g wet. weight) and glutathione peroxidase (GPx) (L: 0.2438+/-0.05 vs 0.0046+/-0.0005; K: 0.1459+/-0.034 vs 0.0055+/-0.0003 U/mg protein). There was no change in catalase and reduced glutathione (GSH) but superoxide dismutase (SOD) increased significantly (P<0.01) (L: 5.49+/-0.76 vs 18.38+/-2.26; K: 11.47+/-1.48 vs 21.22+/-3.19 U/mg protein). Both 500 and 1000 mg/kg/day doses significantly (P<0.05) reduced endogenous antioxidants (catalase and SOD) without altering TBARS. A 1000 mg/kg/day dose of garlic caused marked histopathological and ultrastructural changes in both liver and kidneys. The results suggest that garlic in low doses has the potential to enhance the endogenous antioxidant status, although at higher doses a reversal of these effects is observed. The present study also highlights the potential ability of a high dose of garlic to induce morphological changes in the liver and kidneys, indicating the need to identify a safe dose range for garlic.

Administration, Oral↗
PubMed2000

Ligand binding in the ferric and ferrous states of Paramecium hemoglobin.

The unicellular protozoan Paramecium caudatum contains a monomeric hemoglobin (Hb) that has only 116 amino acid residues. This Hb shares the simultaneous presence of a distal E7 glutamine and a B10 tyrosine with several invertebrate Hbs. In the study presented here, we have used ligand binding kinetics and resonance Raman spectroscopy to characterize the effect of the distal pocket residues of Paramecium Hb in stabilizing the heme-bound ligands. In the ferric state, the high-spin to low-spin (aquo-hydroxy) transition takes place with a pK(a) of approximately 9.0. The oxygen affinity (P(50) = 0.45 Torr) is similar to that of myoglobin. The oxygen on- and off-rates are also similar to those of sperm whale myoglobin. Resonance Raman data suggest hydrogen bonding stabilization of bound oxygen, evidenced by a relatively low frequency of Fe-OO stretching (563 cm(-1)). We propose that the oxy complex is an equilibrium mixture of a hydrogen-bonded closed structure and an open structure. Oxygen will dissociate preferentially from the open structure, and therefore, the fraction of open structure population controls the rate of oxygen dissociation. In the CO complex, the Fe-CO stretching frequency at 493 cm(-1) suggests an open heme pocket, which is consistent with the higher on- and off-rates for CO relative to those in myoglobin. A high rate of ligand binding is also consistent with the observation of an Fe-histidine stretching frequency at 220 cm(-1), indicating the absence of significant proximal strain. We postulate that the function of Paramecium Hb is to supply oxygen for cellular oxidative processes.

Amino Acid Sequence↗
PubMed2000

Targeted localized degradation of Paired protein in Drosophila development.

BACKGROUND: Selective spatial regulation of gene expression lies at the core of pattern formation in the embryo. In the fruit fly Drosophila, localized transcriptional regulation accounts for much of the embryonic pattern. RESULTS: We identified a gene, partner of paired (ppa), whose properties suggest that localized receptors for protein degradation are integrated into regulatory networks of transcription factors to ensure robust spatial regulation of gene expression. We found that the Ppa protein interacts with the Pax transcription factor Paired (Prd) and contains an F-box, a motif found in receptors for ubiquitin-mediated protein degradation. In normal development, Prd functions only in cells in which ppa mRNA expression has been repressed by another segmentation protein, Even-skipped (Eve). When ppa was expressed ectopically in these cells, Prd protein, but not mRNA, levels diminished. When ppa function was removed from cells that express prd mRNA, Prd protein levels increased. CONCLUSIONS: Ppa co-ordinates Prd degradation and is important for expression of Prd to be correctly localized. In the presence of Ppa, Prd protein is targeted for degradation at sites where its mis-expression would disrupt development. In the absence of Ppa, Prd is longer-lived and regulates downstream target genes.

Amino Acid Sequence↗
PubMed2000

Effect of Adriamycin on the boundary lipid structure of cytochrome c oxidase: pico-second time-resolved fluorescence depolarization studies.

The fluorescence dynamics of the dye 3,3'-diethyloxadicarbocyanine iodide (DODCI) was used to probe the microenvironment of cytochrome c oxidase (CcO) and cardiolipin. The dye was partitioned between an aqueous and a hydrophobic phase. The 'bound' and 'free' populations of DODCI could be separated by analysis of the time-resolved fluorescence decay of the dye. The anisotropy decay of the DODCI bound to CcO showed a unique 'dip and rise' shape that was analyzed by a combination of rotational correlation times with time-dependent weight factors for each lifetime component. Rotational dynamics studies revealed the existence of a restricted motion of the dye bound at the enzyme surface. Adriamycin, an anticancer, albeit cardiotoxic drug, was previously proposed to affect the surface structure of CcO, most likely by causing a disorder to the surface lipid arrangement. A drastic change in the rotational correlation time of the dye bound to the enzyme surface was observed, which suggested a depletion of cardiolipin layer due to complexation with the drug. The effect of Adriamycin on cardiolipin was drastic, leading to its phase separation. The present study suggests that the effect of Adriamycin on CcO is primarily a segregation of the cardiolipins.

Algorithms↗
PubMed2000

Mutations in the putative H-channel in the cytochrome c oxidase from Rhodobacter sphaeroides show that this channel is not important for proton conduction but reveal modulation of the properties of heme a.

As the final electron acceptor in the respiratory chain of eukaryotic and many prokaryotic organisms, cytochrome c oxidase catalyzes the reduction of oxygen to water, concomitantly generating a proton gradient. X-ray structures of two cytochrome c oxidases have been reported, and in each structure three possible pathways for proton translocation are indicated: the D-, K-, and H-channels. The putative H-channel is most clearly delineated in the bovine heart oxidase and has been proposed to be functionally important for the translocation of pumped protons in the mammalian oxidase [Yoshikawa et al. (1998) Science 280, 1723-1729]. In the present work, the functional importance of residues lining the putative H-channel in the oxidase from Rhodobacter sphaeroides are examined by site-directed mutagenesis. Mutants were generated in eight different sites and the enzymes have been purified and characterized. The results suggest that the H-channel is not functionally important in the prokaryotic oxidase, in agreement with the conclusion from previous work with the oxidase from Paracoccus denitrificans [Pfitzner et al. (1998) J. Biomembr. Bioenerg. 30, 89-93]. Each of the mutants in R. sphaeroides, with an exception at only one position, is enzymatically active and pumps protons in reconstituted proteoliposomes. This includes H456A, where in the P. denitrificans oxidase a leucine residue substituted for the corresponding residue resulted in inactive enzyme. The only mutations that result in completely inactive enzyme in the set examined in the R. sphaeroides oxidase are in R52, a residue that, along with Q471, appears to be hydrogen-bonded to the formyl group of heme a in the X-ray structures. To characterize the interactions between this residue and the heme group, resonance Raman spectra of the R52 mutants were obtained. The frequency of the heme a formyl stretching mode in the R52A mutant is characteristic of that seen in non-hydrogen-bonded model heme a complexes. Thus the data confirm the presence of hydrogen bonding between the heme a formyl group and the R52 side chain, as suggested from crystallographic data. In the R52K mutant, this hydrogen bonding is maintained by the lysine residue, and this mutant enzyme retains near wild-type activity. The heme a formyl frequency is also affected by mutation of Q471, confirming the X-ray models that show this residue also has hydrogen-bonding interactions with the formyl group. Unlike R52, however, Q471 does not appear to be critical for the enzyme function.

Alanine↗
PubMed2000

Origin of the anomalous Fe-CO stretching mode in the CO complex of Ascaris hemoglobin.

We report an unusually high frequency (543 cm(-)(1)) for an Fe-CO stretching mode in the CO complex of Ascaris suum hemoglobin as compared to vertebrate hemoglobins in which the frequency of the Fe-CO mode is much lower. A second Fe-CO stretching mode in Ascaris hemoglobin is observed at 515 cm(-1). We propose that these two Fe-CO stretching modes arise from two protein conformers corresponding to interactions of the heme-bound CO with the B10-tyrosine or the E7-glutamine residues. This postulate is supported by spectra from the B10-Tyr --> Phe mutant in which the 543 cm(-1) line is absent. Thus, a strong polar interaction, such as hydrogen bonding, of the CO with the distal B10 tyrosine residue is the dominant factor that causes this anomalously high frequency. Strong hydrogen bonding between O(2) and distal residues in the oxy complex of Ascaris hemoglobin has been shown to result in a rigid structure, rendering an extremely low oxygen off rate [Gibson, Q. H., and Smith, M. H. (1965) Proc. R. Soc. London B 163, 206-214]. In contrast, the CO off rate in Ascaris hemoglobin is very similar to that in sperm whale myoglobin. The high CO off rate relative to that of O(2) in Ascaris hemoglobin is attributed to a rapid equilibrium between the two conformations of the protein in the CO adduct, with the off rate being determined by the conformer with the higher rate.

Animals↗
PubMed2000

Redox-linked conformational changes in bovine heart cytochrome c oxidase: picosecond time-resolved fluorescence studies of cyanide complex.

Picosecond time-resolved fluorescence studies are carried out on cyanide-inhibited and heat-modified cytochrome c oxidase in aqueous lauryl maltoside surfactant solution, as well as in an aqueous vesicle, to understand the conformational changes associated with electron transfer and proton pumping activity of the enzyme. The tryptophan fluorescence decay profiles follow a four exponential model, which also matches the lifetime maxima obtained in a maximum entropy method analysis. The fast lifetime components are highly affected by the reduction and chemical modification of the enzyme. Changes in these lifetime components are related to the conformational changes in the vicinity of the heme centers of the enzyme. The cyanide-inhibited enzyme in the oxidized form shows a fluorescence decay profile similar to that of the native oxidized form, indicating that the conformational changes due to cyanide binding are very small. However, reduction of the cyanide-inhibited enzyme that leaves cyanide bound heme alpha3 oxidized causes a large increase in the fluorescence lifetimes, which indicates very significant conformational changes due to electron transfer to the dinuclear Cu(A) and heme alpha centers. A comparison of the tryptophan fluorescence decay of various other modified forms of the enzyme leads us to propose that the possible site of conformational coupling is located near heme alpha instead of the binuclear heme alpha3-Cu(B) center.

Animals↗
PubMed2000

Structural investigations of the hemoglobin of the cyanobacterium Synechocystis PCC6803 reveal a unique distal heme pocket.

A putative hemoglobin (Hb) gene, related to those previously characterized in the green alga Chlamydomonas eugametos, the ciliated protozoan Paramecium caudatum, the cyanobacterium Nostoc commune and the bacterium Mycobacterium tuberculosis, was recently discovered in the complete genome sequence of the cyanobacterium Synechocystis PCC 6803. In this paper, we report the purification of Synechocystis Hb and describe some of its salient biochemical and spectroscopic properties. We show that the recombinant protein contains Fe-protoporphyrin IX and forms a very stable complex with oxygen. The oxygen dissociation rate measured, 0.011 s(-1), is among the smallest known and is four orders of magnitude smaller than the rate measured for N. commune Hb, which suggests functional differences between these Hbs. Optical and resonance Raman spectroscopic study of the structure of the heme pocket of Synechocystis Hb reveals that the heme is 6-coordinate and low-spin in both ferric and ferrous forms in the pH range 5.5-10.5. We present evidence that His46, predicted to occupy the helical position E10 based on amino-acid sequence comparison, is involved in the formation of the ferric and ferrous 6-coordinate low-spin structures. The analysis of the His46Ala mutant shows that the ferrous form is 5-coordinate and high-spin and the ferric form contains a 6-coordinate high-spin component in which the sixth ligand is most probably a water molecule. We conclude that the heme pocket of the wild type Synechocystis Hb has a unique structure that requires a histidine residue at the E10 position for the formation of its native structure.

Amino Acid Sequence↗
PubMed2000

Characterization of a two-component system, devR-devS, of Mycobacterium tuberculosis.

By subtractive hybridization, we isolated genes, differentially expressed in virulent strain (dev), that are expressed at higher levels in the virulent Mycobacterium tuberculosis H37Rv strain in comparison to its avirulent counterpart, H37Ra, and consequently may be associated with the virulence phenotype of M. tuberculosis. A two-component system, devR-devS, was identified by DNA sequencing of a dev clone. DevR, the predicted gene product of devR, is a response regulator (RR) in the NarL/ UhpA subfamily of two-component systems. The devS gene product displayed homology with histidine protein kinases (HPKs) including UhpB, NarX and NarQ. The devR-devS locus is preceded by gene Rv3134c that encodes a putative alanine-aline- rich protein. This locus was conserved in M. tuberculosis and M. bovis BCG but not in other mycobacteria. A devR -lacZ transcription fusion demonstrated beta-galactosidase activity in M. smegmatis and in M. tuberculosis. The devR and devS genes were cotranscribed and the levels of their transcripts were lower in two isolates of the avirulent H37Ra strain in comparison to the virulent H37Rv strain of M. tuberculosis. The level of DevR protein was also lower in one of the H37Ra strains in comparison to the H37Rv strain. However, in a third isolate of H37Ra, RNA and protein expression was equivalent to that in the H37Rv strain. Electron microscopic immunogold analysis of M. tuberculosis grown in laboratory medium and within human monocytes revealed specific labelling for DevR protein within the bacteria and the phagosomal lumen of infected monocytes. These findings collectively suggest a potential role for devR-devS in the regulation of genetic programmes unique to the tubercle bacillus.

Cells, Cultured↗
PubMed2000

Etiology based prevalence of Budd-Chiari syndrome in eastern India.

BACKGROUND: Diagnosis of Budd-Chiari syndrome (BCS) is often missed unless its possibility has been kept in mind. Obstruction of inferior vena cava (IVC) is reportedly the most frequent cause of BCS in Afro-Asian variety. AIM: An attempt was made to classify BCS (in an eastern Indian population) etiopathologically. PATIENTS AND METHODS: Thirty consecutive cases of BCS presenting over a period of five years were included. Following a thorough physical examination, necessary investigations (including coagulation profile, ultrasonography (with Doppler study) of hepatobiliary tract, hepatic vein and IVC angiography (n = 22) and liver biopsy (n = 26, including autopsy in two cases) were performed. RESULTS: Mean age at presentation was 32.7 +/- 10.36 years (range 12-60 years) with M:F = 21:9. Clinical presentations included, hepatomegaly in 28 (93.3%), ascites in 27 (90%), splenomegaly in 15 (50%), pain in abdomen in 26 (86.6%), jaundice in 10 (33.3%), back veins in 20 (66.6%) and gastrointestinal bleeding in three (10%) cases. Amongst the total of 30 patients, four, six and 20 cases presented as fulminant, acute and chronic BCS respectively. Twenty four cases of BCS could be diagnosed by ultrasonography alone, while the remainder required angiography for diagnosis. IVC and hepatic vein angiography revealed membranous obstruction in nine, partial stricture of IVC in six, and IVC and/or hepatic vein block in others. The etiopathological nature in 30 cases were as follows: idiopathic membranous obstruction in nine (30%), hepatocellular carcinoma in six (20%), idiopathic stricture in six (20%) cases and one case (3.3%) each of the following: cholangiocarcinoma, renal cell carcinoma, chronic pancreatitis, hydatid cyst in liver, protein S deficiency, oral contraceptive use, nephrotic syndrome (with antithrombin III deficiency), polycythemia rubra vera and chronic lymphatic leukemia. CONCLUSION: Idiopathic membranous obstruction and stricture of IVC are the commonest cause of BCS in the eastern part of India. Hepatocellular carcinoma is also a common cause, presenting in the fulminant form. Ultrasonography may be a helpful screening test for BCS, but IVC and hepatic vein catheterisation is essential for a complete work up of these patients.

Adolescent↗
PubMed1999

Identification of the ligands to the ferric heme of Chlamydomonas chloroplast hemoglobin: evidence for ligation of tyrosine-63 (B10) to the heme.

We have studied the unusual heme ligand structure of the ferric forms of a recombinant Chlamydomonas chloroplast hemoglobin and its several single-amino acid mutants by EPR, optical absorbance, and resonance Raman spectroscopy. The helical positions of glutamine-84, tyrosine-63, and lysine-87 are suggested to correspond to E7, B10, and E10, respectively, in the distal heme pocket on the basis of amino acid sequence comparison of mammalian globins. The protein undergoes a transition with a pK of 6.3 from a six-coordinate high-spin aquomet form at acidic pH to a six-coordinate low-spin form. The EPR signal of the low-spin form for the wild-type protein is absent for the Tyr63Leu mutant, suggesting that the B10 tyrosine in the wild-type protein ligates to the heme as tyrosinate. For the Tyr63Leu mutant, a new low-spin signal resembling that of alkaline cytochrome c (a His-heme-Lys species) is resolved, suggesting that the E10 lysine now coordinates to the heme. In the wild-type protein, the oxygen of the tyrosine-63 side chain is likely to share a proton with the side chain of lysine-87, suggested by the observation of a H/D sensitive resonance Raman line at 502 cm(-)(1) that is tentatively assigned as a vibrational mode of the Fe-O bond between the iron and the tyrosinate. We propose that the transition from the high-spin to the low-spin form of the protein occurs by deprotonation and ligation to the heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.

Amino Acid Substitution↗